Week 5

 First off, I would like to apologize for not posting earlier and for not posting last week. I had a little issue locating the blog. I have two Google accounts so I would sign in with my other account onto blogger and the blog would not appear. Now that I had some time to think about and solve the issue, I have located the blog in my other account as you guys probably figured out if your reading this. Anyways now for the science...

      In week five we have finally acquired enough Bid protein to run the NMR! Like, anything else we have done in Pellecchia's Lab, they want us to acquire the necessary background and knowledge to be successful in an experiment and to give us an idea of why and how things happen. Saying thus, we were given a number of articles on NMR, which included how it works, what region to look at in our experiment, the advantages, disadvantages, etc. After going through the articles we briefly helped Elisa (our post-doc) preparing the NMR tubes to get ready for the screening. It was delayed because both of the NMR machines were down.  She also explained to us the project much more in-depth and what exactly are we looking for. In the mean time while the delay we started to express EphA3, once again.

    The second presentation was also held in week 5. I believe everyone has encountered their share of successes and failure, which is nice to sit down and talk about the failures as much as the successes. I felt everyone more relaxed, during the presentations because we knew what to expect. Overall, I believe everyone did a good job and just need to continue implementing  everyone's advice and continue working hard and with a purpose.

 

My Experience as an Intern week 5

     This was a great week and a not so great week. Elisa was able to use the protein we purified to run a 1D HSQC NMR experiment of our protein. From the spectra we can tell that there is nothing wrong with our protein it is well folded. Elisa one of our lab mentors, has a sample of very precious protein that she needed to screen, and was just sitting in 4℃. Jose and I were going to learn how to set up the sample for NMR analysis, unfortunately, one of the NMR machines was not working. The second machine was working there were some issues with the sample changer but Elisa said we could do this manually.  Then, sadly there were some issues with the sample changer and Elisa could not start the experiments over the weekend.

     In regards to protein expression, Jose and I started to express the second protein of interest. Unfortunately, we did not get good purification or yield. We are going to give it a second attempt.  Along with the laboratory work, we have also been learning background information necessary for NMR use, and understanding. Elisa is great mentor and does a great job simplifying the concepts for us.

     I really enjoyed learning about all of your advancements and struggles in your respective experiments. It is great to hear about what we all do. I wish you all great luck in your experiments and I am excited to hear more about what your findings are. Wish you all have a great week  J .

week 5

Last week was a busy week, one of my priorities was to obtain data from our Dot Blots so that Claudia and I could have something to present on Friday. The main focus of the week was understanding Dot Blots, learning how to performed a Dot Blot, and determining the right concentration of antibodies use for the Pla Assay. Although we didn't get to scan our Dot Blot with HNE we still got good data which we presented on our Friday.

Week 5

During the week I met with Chuck in order to go over some of my data and my approach to analysis. After talking with Chuck and determining what type of approach I would take in data analysis, I quickly saw that there was not enough data available for the specific regions I had in mind. Thus, as I stated in the presentation this presented itself as a limitation for my project.

After presenting on Friday I received quite a bit of helpful questions and suggestions. As suggested, I will now try to look at obesity over 5 year increments (of the data is available) for all California counties instead of just San Joaquin valley. Thus, I will be attempting to do a comparison of the specified variables between different regions or groups of counties. In doing this I will be able to see if there is a trend and better establish whether or not there is a relationship between obesity and prostate cancer.

Another insightful suggestion I received was to look at whether there was more than just a linear relationship between the two variables. Obesity is a complex disease and it is likely that the relationship with cancer (if it exist) is more complicated than thought to be. I will continue to meet with my mentors and see if I can establish a better plan of analysis and hopefully depict clearer and more in depth results.


Overall, it was interesting to see how everyone's projects are going along. It was also a relief to hear that others were experiencing some difficulties as there projects progressed as well.

Week 5

      This week was particularly interesting. We spent the week without Judith or Dieter in the lab all the time. We performed a couple western blots. It was different working independently. Since we didnt get any definitive results, I question whether I did each part properly. I appreciate the suggestion to use the control to see if there were any error in the procedure.

  This weeks presentations also went by smoothly. It seemed like everyone was much more relaxed since they knew what exactly to expect. The updates on the projects were informative.

Finally, today, Jose, Luis and I went to Rock Bottom to watch the final of the Gold Cup. It was a fun and good time. We dont have espn so it was fun to watch sports on the big screen.

That's all for this week.

Week 4

Hello everyone, on week4, we were not able to commence our PLA due to time constraints. My thoughts for week four were, it would be a very busy week, lab wise, but as you have read it was not the case, because we only had two full work days. This does not mean we were not busy, because we were, but doing other things. Here is what we did, we attended Dr. Salvesen’s talk that was given to the Summer Bridge students at UCSD on Monday July, 15. I really liked the way Dr. Salvesen presented on apoptosis. Specially how he gave names to the proteins depending on the role they have on the pathway. It was the first time I heard the proteins be called jury, judge and executioners but it reinforce his explanation of the apoptotic pathway. All of the six interns worked on a presentation we did for Journal club at UCSD for the Bridge program. In addition, we had the opportunity to attend the 2013 Cancer Center Post-doc/Graduate student retreat. I had a great time at the retreat. I enjoyed learning what other types of research is taking place at Sanford Burnham’s Cancer Center. In addition, it was very interesting to see the graduate students presenting styles. Some of the presentations were added to some of the best presentations I have seen in my life. I really enjoyed the presenters that made their talks into a story. They had very good transitions from one slide to the next or from one experiment to the other. They made it clear that you need to have many experiments to test your hypothesis. Finally but not least, Luis and I got to take a look at protocols of the techniques we will be using for Week 5.

week 4

Last week was a very slow week because we did not get to do much lab work due to events that took place outside the lab. On Monday we attended a seminar on programmed cell death given by Dr. Guy Salvesen which helped me better understand the Neutrophil project. Then on Wednesday we had to review an article on cell death for the students in Bridge program. On Thursday we attended postdoctoral retreat in which graduates students and research professionals gave presentations on the projects that they were working on. It was a great learning experience watching them present their data and I plan to use what I learned on my future presentations.

Week 4

This week was very different from the weeks prior.

First, we presented an article by Dr. Salveson et al that discusses the activation of the apical caspases. We were able to split the presentation between the 6 of us. I think it went really well. A few people asked questions, and more were baffled that we found some problems with the science (more so the presentation of) in the paper. Afterward, a couple of the Bridge program organizers commended us on a job well done.

Secondly, we attended a retreat/ symposium on Thursday. We saw many presentations by grad students and post-docs. I found it a bit hard to follow at time since they used advanced terminology. Overall, I thought it was an in depth look at the lab experience of PhD/post-doc. During lunch, a couple of us met Dr. Ze'ev and Bill. With Dieter (Dr. Wolf wants us to address him as Dieter), they discussed the perils and problems of acquiring funding and maintaining a lab. The sandwiches served at lunch had interesting combinations of flavors; roast beef with a weird horseradish and turkey with a cranberry jelly.

This was our first full week with Dieter in lab. He helped us perfect our PCR technique and troubleshoot it. On Friday, we ran a gel to examine our PCR from Wednesday, and IT WORKED! This next wekk will be interesting since both Dieter and Judith will be out of lab. We will be running western blots and PCRs to look for different things.

Lastly, I went exploring on Saturday. I grew weary of hanging out in my room, so I walked around the village east. There are some cool terraces on the 12th floor ( or on top of one of the ecoflats building) where you can relax. I spent a couple hours reading my book. On Saturday, I also adventured to Serra Mesa where I got a haircut at one of the oldest barbershops in the area.

This is all for now. I will check in again soon!

July 19

Jose and I just met with Dr. Kwon yesterday afternoon and we discussed once again our data research. We had been looking through SEER at the various data sets available in regards to breast cancer and prostate cancer. Looking at county level and the hispanic population in particular in comparison to other groups showed that the white race presents both higher incidence and mortality rates. Dr. Kwon then suggested we take a different approach and focus on the risk factors of different groups. Another interesting observation was that there was a clear decrease of incidence and mortality rates over time which is great! But it is also interesting to look into the possible diagnoses or treatment that have made this possible. There is also disparity on genetics that could be approached in our study and further research will be conducted to find studies that are focused on this particular area.

Week 3 Experience

My week three experience in the lab was definitely a new and exciting learning process. I can say the researching and preparation, before the actual protein expression was definitely helpful. It gave me a great insight on the whole process and the science of why things are done and happen in the expression. Since our post-doc mentor was on vacation, we were paired with John Stebbins, a fellow scientist in the lab. He was definitely helpful and showed and explained each step of the protein expression. Our results were not what we expected, as we did not obtain a high protein yield. We will re-try the expression next week on our own. I definitely feel prepared and excited to re-do the expression without any major assistance besides some questions we may have during the expression.

Week 3 also consisted of our first Fresno-Sanford Burnham journal conference. I felt that the presentations were good, overall. A lot of great suggestions were said, that I believe should be inputted into future presentations. I definatley felt prepared and comfortable for the presentation, as we had practice runs and advice from Dr.Pellecchia and John. I look forward to starting the protein expression next week on our own. 

My experience as an intern week 4

          Jose and I were left on our own. We were to start the protein expression protocol on our own with out major guidance from the scientist. If we had any questions we could freely ask and were given a response. We were bothy really enthusiastic about running the autoclave and setting up our media to start cultures to work with today. To our dismay, the autoclave was not working :( Jose and I had our day all planned. The scenario reminded me that things especially in biology do not always go as expected. The nonfunctional autoclave would move us a bit behind of our planned out schedule. Luckily, after attending Dr. Salvesen's talk and then meeting with the rest of the cohort (to dissect the journal article we will be presenting for journal club at UCSD) we went to the autoclave and a kind person had already placed our flask in the autoclave. Our day was saved by this act of kindness and we successfully started the cultures.

My experience as an intern during week 3

          My third week working in the lab was great. I was happy to start working and getting my hands on the pipetters. We were introduced to the technique optimized for the expression of the protein in E. coli BL21 starr cells to grow batches of bacteria. I enjoyed seeing how the equipment at the institute has facilitated the smooth progress of experiments. Personally, I was thrilled to learn a new technique that assist in lysing cells open. There is a pressure homogenizer to lyse cells open, never before had I seen one, since I am most familiar with the use of the sonicator. We also worked on our first presentation related to the project. Dr. Pellecchia even gave us feed back via e-mail. It was great to get suggestions from abroad. As soon as we completed the expression of the protein of interest we ran the sample on an SDS PAGE. Unfortunately, our protein expression did not go s planned, we did not get the results we expected.

          Presentations went well. Personally, both Jose and I identified some areas of improvement and got some ideas from the other presenters. In respect to the other presenters, I just have some comments that I think will help the audience understand the information. Some of the explanations should be somewhat simpler and please label graphs, charts and diagrams. Some slides did not contain titles on their figures or axis and this was somewhat confusing. Including this sort of information would really come handy, because the audience will have more time to focus on what you are delivering instead of attempting to decipher what the graphs are representing.

Week 3

Hi everyone, I really enjoyed learning about what you all were working on for the past three weeks. I look forward on learning more on everyones research and see what their outcome is. So this past week, Luis and I continue to do Kinetics with the assistance of Dr. Salvesen and Scott. The process we went through to obtain good results was interesting and a great learning opportunity. Luis and I found out that data obtained from kinetics can be misleading because not all data obtained is what it appears to be. We got data that looked like it was good but in reality it did not graph a nice Michaelis Menten or a linear graph. Eventually, after Scott’s and Dr. Salvesen’s troubleshooting we were able to get good results by lowering the substrate concentration. So as all of you have learned, this week (week four) we will be  working on NETs and PLA and I am super excited.  

Week 3

This week went by fast and the presentation on Friday only added more pressure for Claudia and I to complete our Enzyme kinetics so that we could have data to present on Friday. I was not happy with the presentation we gave on Friday and I know that it could have been better. I have learned a lot from my experience this week and I will prepare myself better for the next presentation.

Week 3

     This week went by really quickly. In lab I learned a few more techniques. We ran polysome profiles, and made the gradients that go with them. On the profile, each peak corresponds to part of the lysate of the cells. The first big peak is the lysate which is full of extra protein and light cell parts. After that, we have peaks for the 40s and 60s subunits of the ribosome. The 80s comes next. Following the 80s, we come to the polysomes ( strands of mRNA with multiple ribosomes actively elongating). The higher the peak the more of the respective substance is in that layer. It has been observed that when eIF3e is deleted we see fewer high number polysomes ( polysomes of 3 and higher).

     This week I finally got to meet Dr. Wolf. He had been out of the country for the past few weeks. He helped us optimize our PCR. We ran our DNA again and still have to interpret the images from the gel.

     On Tuesday, we had dinner downtown for Jose's birthday. Trying to find parking was awful. Luckily, I found a garage that wasn't too expensive nor was it too far away. For dinner, we ate at Yardhouse which was an interesting restaurant. Since we don't ESPN in our apartment, it was nice to be able to watch some sports again.

    Lastly, we had our first presentation this week. I think it went really well for everyone. It was interesting hearing about the projects and hearing the PIs' various questions. It was particularly entertaining to see Dr. Wolf and Dr. Salveson go back and forth discussing the details of the Salveson project.

Week 2

As everyone probably mentioned, this week was a short one due to the holiday. Although it was a short week, Luis and I were as busy as last week. We got to run several SDS-Page gels. In addition we were able to learn and shadow the rest of the steps needed for a western blot. We also learned more about our project and did some enzyme kinetics, which we will continue to do on week three.

My experience as an intern during week 2

As a result of the July 4 holiday, our time spent in lab during week #2 was only three days, during which Jose and I continued to learn about the project we will be tackling for the remainder of our time here at the institute. Most of our time was spent working on our power point presentation. We also had two meetings with Dr. Pellecchia. He introduced the new protein EphA3, that he and his lab members are interested in studying further. He gave us a brief overview of the overall aim of the project and indicated what direction the project is headed to. I am thrilled to start actual experiments on Monday. I will definitely keep you all posted on our experience in the bench. A postdoc will be assisting us with protein expression of Bid. Jose and I will be busy bees for the duration of this third week :). I am filled with excitement to start working and I can't wait to see how the project progresses as the weeks go by, especially since I will be learning about NMR. The application of this technique is new to me and I am really excited to add it to my tool box at the end of the summer :)

Week 2

          Another week has past us by at Sanford Burnham and I have to say I am really becoming comfortable with our project, which deals with the protein Bid and EphA3. Our goal is to identify and possibly synthesize peptide(s) that bind to Bid. We plan to do this by first purifying and expressing the protein, screening the library of compounds by NMR, synthesizing the individual peptides, and finally characterizing the peptides. Through this point, we have been detailing our course of action, mapping out our deadlines for our eight weeks here,  intensively researching and gaining more background knowledge for our project and formulating our presentation for this upcoming week. Since our post-doc mentor,  Elisa is on vacation, we have been able to interact with Dr. Pellechia more on a daily basis. He has clearly mapped out and explained his expectations and has also been a big help in regards to our presentation by giving us pointers and constructive criticism. I am actually pretty excited to present! This upcoming week is when the real fun starts as we will be commencing our protein expression of Bid!

Week 2

     Since there are holidays for the 4th and 5th, my second week in lab has come to a close.

     This week we mainly ran PCRs for ssa2 and sks2. Unfortunately, none of the gels have given us results. Subsequently, we have spent time trying to troubleshoot our methods. We have tried a rerunning the samples (while being more meticulous), running the samples on a temperature gradient, trying samples with new forward and reverse primers, and finally trying the sample on a different machine. Since none of those improved the images we got, Judith recommended that we try using different concentrations of the parts of the PCR (eg less concentrated genomic DNA).

     Fortunately, I have become much more comfortable with making the gels, performing the electrophoresis, and imaging the gels. Judith is an excellent teacher and is always there to help me improve my techniques, expand my knowledge, and answer all of my questions.

   Finally, we ran another part of our western blot. We added rabbit antibodies to get a darker result. Judith explained that the rabbit antibodies are not as specific as the ones we used previously (the rat), and it showed on the picture. There was a significant increase in the unspecific binding, and the image was "dirtier" (the image was darker and there were many sections with a dark haze).

   This week I started commuting by bike to Sanford-Burnham. It is much faster than walking. That is unless you get a flat tire which happened when I tried to return today. It was a lovely walk with my bike on my back!

    I'm looking forward to next week and learning more. So far, it has been very educational and a lot of fun. Judith is a patient and great teacher! I am starting to feel much more comfortable with the pipetting, gel electrophoresis, and the other procedures that we performed.

Hard at work in the Salvesen lab

Kinetics

Claudia and Luis hard at work on enzyme kinetics, with Scott Snipas.  Everyone is still smiling!

Naturally the first experiments conducted by Scott and Guy Salvesen went a bit wrong, but a swift change of buffers, and the capable hands and minds of Claudia and Luis saved the day, just in time for the holiday!